Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transitions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic (P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observable ranged from 0.06 (P. vittata) to 0.12 (P. setiferum)gH(2)Og(-1)dry weight, and were highly correlated with water affinity parameters. In spores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum)gH(2)Og(-1)dry weight, some water partitioned into a near pure water fraction that melted at about 0 degrees C. These sharp peaks near 0 degrees C were associated with lethal freezing treatments. The enthalpy of water melting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores compared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fern spores, water content can be precisely manipulated to avoid both desiccation and freezing damage.     

Effects of temperature and desiccation on ex situ conservation of nongreen fern spores

? Premise of the study: Fern spores are unicellular and haploid, making them a potential model system to study factors that regulate lifespan and mechanisms of aging. Aging rates of nongreen spores were measured to compare longevity characteristics among diverse fern species and test for orthodox response to storage temperature and moisture. ? Methods: Aging of spores from 10 fern species was quantified by changes in germination and growth parameters. Storage temperature ranged from ambient room to -196°C (liquid nitrogen); spores were dried to ambient relative humidity (RH) or using silica gel. ? Key results: Survival of spores varied under ambient storage conditions, with one species dying within a year and two species having greater than 50% survival after 3 years. Few changes in germination or growth were observed in spores stored at either -80°C or -196°C over the same 3-yr study period. Spores stored at -25°C aged anomalously quickly, especially those dried to ambient RH or subjected to repeated freeze-thaw cycles. ? Conclusions: Spore longevity is comparable to orthodox seed longevity under ambient storage conditions, with wide variation among species and shelflife extended by drying or cooling. However, faster aging during freezer storage may indicate a similar syndrome of damage experienced by seeds categorized as "intermediate". The damage is avoided by storage at -80°C or liquid nitrogen temperatures, making cryoconservation an effective and broadly applicable tool to extend fern spore longevity. The study demonstrates that spore banks are a feasible approach for ex situ conservation of this important plant group.     

Fern and lycopod spores rain in a cloud forest of Hidalgo,Mexico

The aim of this study was to determine the composition of the “spore rain” of ferns and lycopods in a cloud forest. We tested whether the canopy impedes spore dispersal to surrounding areas and how spore dispersal is affected by rainfall. The spores were captured with a modified Bush–Gosling trap placed at 30 cm above ground level in forested and non-forested sites from March 2009 to February 2010. We collected 2462 fern spores from 158 morphospecies of which 76 were identified to species level. Thirty-seven species were found exclusively in the spore rain, and 39 were found as sporophytes as well (local component). Mean daily spore density (spores m^?2) was calculated to find the sporulation period for each species. Twenty species showed seasonal patterns of sporulation. The highest spore density was found at the forested site (70 morphospecies and 1856 spores), of which 39 morphospecies (1482 spores) corresponded to the local vegetation. Fifty-five taxa were shared between the forested and non-forested site. In the non-forested site, 605 spores were captured belonging to 64 species. The density of spore rain between sites was significantly different. The rainfall amount was the same at both sites, with a dry period in March, April, and July 2009, and February 2010. There was a negative effect of rainfall on spore rain. The main sporulation occurred in the dry season with strong winds. Although the canopy inhibits airborne dispersal of fern spores, a small amount of spores can disperse beyond the canopy and reach surrounding areas. The rainfall might wash spores to ground and favor the colonization and the establishment of new populations.     

Fern Spore Longevity in Saline Water: Can Sea Bottom Sediments Maintain a Viable Spore Bank?

Freshwater and marine sediments often harbor reservoirs of plant diaspores, from which germination and establishment may occur whenever the sediment falls dry. Therewith, they form valuable records of historical inter- and intraspecific diversity, and are increasingly exploited to facilitate diversity establishment in new or restored nature areas. Yet, while ferns may constitute a considerable part of a vegetation’s diversity and sediments are known to contain fern spores, little is known about their longevity, which may suffer from inundation and - in sea bottoms - salt stress. We tested the potential of ferns to establish from a sea or lake bottom, using experimental studies on spore survival and gametophyte formation, as well as a spore bank analysis on sediments from a former Dutch inland sea. Our experimental results revealed clear differences among species. For Asplenium scolopendrium and Gymnocarpium dryopteris, spore germination was not affected by inundated storage alone, but decreased with rising salt concentrations. In contrast, for Asplenium trichomanes subsp. quadrivalens germination decreased following inundation, but not in response to salt. Germination rates decreased with time of storage in saline water. Smaller and less viable gametophytes were produced when saline storage lasted for a year. Effects on germination and gametophyte development clearly differed among genotypes of A. scolopendrium. Spore bank analyses detected no viable spores in marine sediment layers. Only two very small gametophytes (identified as Thelypteris palustris via DNA barcoding) emerged from freshwater sediments. Both died before maturation. We conclude that marine, and likely even freshwater sediments, will generally be of little value for long-term storage of fern diversity. The development of any fern vegetation on a former sea floor will depend heavily on the deposition of spores onto the drained land by natural or artificial means of dispersal.     

Fern and bryophyte endozoochory by slugs

Endozoochory plays a prominent role for the dispersal of seed plants, and dispersal vectors are well known. However, for taxa such as ferns and bryophytes, endozoochory has only been suggested anecdotally but never tested in controlled experiments. We fed fertile leaflets of three ferns and capsules of four bryophyte species to three slug species. We found that, overall, spores germinated from slug feces in 57.3 % of all 89 fern and in 51.3 % of all 117 bryophyte samples, showing that the spores survived gut passage of slugs. Moreover, the number of samples within which spores successfully germinated did not differ among plant species but varied strongly among slug species. This opens new ecological perspectives suggesting that fern and bryophyte endozoochory by gastropods is a so-far-overlooked mode of dispersal, which might increase local population sizes of these taxa by spore deposition on suitable substrates.     

Evidence for natural selection in a fern hybrid zone

Morphological characters, chloroplast DNA, and allozymes were used to analyze the distribution of individuals within a hybrid population of the ferns Polystichum munitum and P. imbricans in northwestern California. Microsites within the population were characterized according to soil moisture and light levels reaching the plants. In sites with low soil moisture and high light levels, all of the ferns were genetically and morphologically like P. imbricans. In contrast, ferns with the genetic and morphological identity of P. munitum predominated in moist shady sites. Intermediate sites supported very few P.munitum, a wide variety of hybrid recombinants, and a majority of ferns with P. imbricans characteristics. The pattern of variation within the population is noteworthy because of the close proximity of the habitat extremes and the long-range dispersal of fern spores. We conclude that natural selection along environmental gradients must be a major factor in determining the ecological and genetic associations within the hybrid zone. The results of this study are evaluated in the context of the fern life cycle and compared to the assumptions of models explaining the establishment and maintenance of hybrid zones, which vary in the role attributed to environmentally mediated natural selection.     

Fern-spore-feeder interaction in temperate forests in Japan: Sporing phenology and spore-feeding insect community

Angiosperms are widely appreciated to have flowering schedules, but far less attention has been paid to the timing of spore production by ferns. Although a range of abiotic factors are likely responsible for the timing of fern sporing, spore predation by specialist spore-feeding insects may also exert selective forces on timing. As a step toward understanding ecological factors that affect the evolution of fern sporing phenology, we tracked annual sporing patterns and examined associations with spore-feeding insects in 38 ferns species in two Japanese temperate forests. Most sporing occurred during June through August, the period of highest temperature and precipitation at the study sites, but some species produced spores during a limited period in spring or very late autumn. Over 70% of all species examined were attacked by spore-feeders, which consisted of seven polyphagous and five oligophagous species. Spore feeders occurred predominantly during June through September, and stathmopodid moth larvae consumed up to 70% of the mature sporangia. Thus, although the warm and humid conditions in the summer is likely favored for prothallial growth and fertilization, spring or late-autumn sporing in some species may have evolved as an adaptation to escape spore predation by spore feeders.     

Seasonal variation in the spore bank of ferns in grasslands on dolomite rock

Composition and seasonal patterns of the fern spore bank were compared to the surface vegetation of grasslands on dolomite rock in Hungary. Viability and potential dormancy of spores were tested through storage experiments. Although Asplenium ruta-muraria L. was the only species found at the study sites, five others, probably originating from air-borne spores from nearby areas, emerged from the soil samples. Considerable seasonal variability was detected in the number of prothallia emerging from soil samples from different sampling dates, with a peak after spore dispersal. The increased number of emerging prothallia after 1 year of storage suggests that a part of the spores stored in the soil samples were presumably dormant. Investigations on the dormancy of fern spores might be of great interest, especially in species adapted to seasonally unfavourable habitats.     

First report of fern (Culcita macrocarpa) spore consumption by a small mammal (Apodemus sylvaticus)

Few vertebrates are known to consume ferns regularly. Several species of mammals consume leaves to some extent but the consumption of fern spores is much rare. In Galicia (Northwest Spain) we studied the seasonal variation in the consumption of Culcita macrocarpa fertile leaves (i.e. with spores) in two populations (Capelada and Eume), assessed whether consumption rate increased with fern population size, and evaluated whether the consumer was a spore predator or a spore disperser. Consumption began in December and finished by mid February, and occurred before spore release, which happened later in Capelada than in Eume, probably influenced by differences in altitude. The consumer was identified as Apodemus sylvaticus by DNA analysis of its droppings and by capture of live animals. Throughout Galicia there was a significant increase in fern consumption rate as the population size of C. macrocarpa increased. Germination tests from droppings were carried out in 14 dishes but only in two dishes 1% and 0.3% of the spores germinated. Our results suggest that woodmouse can disperse spores of C. macrocarpa, although most of the spores were digested.     

Fern spore fall in the ‘Parque Estadual das Fontes do Ipiranga (PEFI)’, São Paulo,Brazil

Studies related to spore production and dispersal are scarce but extremely important, as they allow colonisation of new sites and gene flow among different populations. The aim of this study was to investigate fern spore fall in the Parque Estadual das Fontes do Ipiranga (PEFI), a natural reserve in the State of São Paulo, Brazil. The analyses were performed every month for one year, using an Oldfield pollen trap, 30 cm above the ground, at four different sites. The method included introducing Lycopodium clavatum tablets, adding hydrofluoric acid and sulphuric acid, and finally adopting the acetolysis method. A total of 17 types of spores were identified. The canonical correspondence analysis (CCA) did not demonstrate regular patterns of dispersal for each site. Wind influences the spore dispersal process minimally, but there was no significant association between dispersal and the remaining environmental variables. Spores of species uncommon to the area were recorded, perhaps contributing to the colonisation of new areas and helping to understand studies on regeneration and recomposition of ferns and lycophytes in areas undergone anthropic impacts.     

液体培养基条件下乌毛蕨配子体发育的研究

在液体培养基条件下对中国产乌毛蕨(Blechnum orientaleL.)配子体发育进行了观察。结果表明:乌毛蕨的孢子为两面体型,两侧对称,极面观为椭圆形,赤道面观为半圆形或豆形,周壁具脊状褶皱;孢子萌发为书带蕨型;原叶体发育为三叉蕨型,成熟原叶体为对称的心脏形。经比较分析,蕨类植物孢子繁殖时采用混合土培养较适宜;液体培养基和混合土在研究蕨类植物配子体发育时同样具有可行性。孢子形态和配子体发育的观察结果表明,乌毛蕨是蕨类植物中较进化的种类;乌毛蕨科与鳞毛蕨科亲缘关系较近。     

Autochory in ferns,not all spores are blown with the wind

Dispersal is a key process in plant population dynamics. In ferns, two successive vectors are needed: the sporangium catapulting mechanism, and wind or gravity. However, some rock ferns have a growth habit that suggests a kind of autochory by placing spores on the rock surface. Moreover, some ferns show modifications of the sporangial dehiscence. To determine the role of growth habit in spore dispersal, we checked the sporangial opening mechanism and explored the spatial distribution of plants on the walls. The presence of spores of Asplenium celtibericum, a rupicolous fern, in the rock surface was checked. In addition, its sporangial dehiscence, plant size and position in the wall were analysed. Spores and indehiscent sporangia were present on walls at each sampling moment. Their highest number was found close to the plants. There was a positive correlation between crack width and plant size. However, most plants occupy the upper half of the cliffs. The growth habit of A. celtibericum is instrumental to deposit the spores over the neighbouring rock surface, thus enhancing the probability of spores to find suitable crevices for germination. Furthermore, dispersal of indehiscent sporangia might promote intergametophytic mating, and the modified sporangial opening mechanism extends the dispersive period.     

Biochemistry of fern spore germination: protease activity in ostrich fern spores

Protease activities were detected in quiescent and germinating spores of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Peak endopeptidase, aminopeptidase, and carboxypeptidase activities were detected 12 to 24 hours after spores began imbibing under light. There was a correlation between activities of proteases, the onset of a decline in levels of soluble protein, and an increase in levels of free amino acids. The earliest visible event of spore germination, breakage of the spore coat and protrusion of a rhizoid cell, was observed after peak protease activity, 48 to 72 hours after the start of imbibition. Results of this study demonstrate similarities in the pattern of protease activities during germination of ostrich fern spores to those of some seeds.     

环境因子对蕨类植物孢子萌发的影响

蕨类植物通过孢子萌发形成独立生活的配子体,配子体能够形成精子器和颈卵器,进而通过受精作用形成新的孢子体。孢子萌发是蕨类植物生活史过程中配子体世代向孢子体世代转变的关键步骤。同时,此过程不仅受到多种环境因子的影响,也是研究细胞核极性移动、细胞不对称分裂、假根极性生长等独特的细胞学事件的良好模型。迄今为止,人们已经研究发现多种环境因子对约200余种蕨类植物孢子萌发有影响。总结了环境因子对蕨类植物孢子萌发影响的规律如下:(1)孢子萌发除了受到光照强度影响外,主要受光质的影响,光质的影响主要表现为4种方式:①孢子萌发受红光刺激与远红光抑制像开关一样调控;②孢子萌发不受远红光抑制;③孢子萌发受蓝光抑制;④孢子只能在黑暗条件下萌发。(2)重力作用会影响孢子细胞核移动,进而影响孢子细胞发育的极性。(3)赤霉素(GA)能增加孢子萌发率或帮助孢子打破休眠。成精子囊素与GA作用相似,启动或促进孢子萌发。而脱落酸(ABA)、茉莉酸(JA)和乙烯等其它激素对孢子萌发的影响相对较小。(4)不同植物孢子有着各自最适的萌发培养基条件,如不同种类孢子对MS培养基中无机盐含量、蔗糖含量、pH值的要求不同。孢子外被中的Ca2+、Mn2+和Mg2+,培养基中的Cd2+和La3+,以及孢子接种密度、萌发空间CO2含量也会对孢子萌发造成影响。(5)多数蕨类植物孢子在15-30℃可以萌发,最适萌发温度为25℃。(6)4℃和液氮储藏可以延长孢子寿命并保持较高萌发率。     

The surface coats on spores

There are surface coats on the sporocytes and spores of some pteridophytes and bryophytes which will bind one or more of the stains generally used to demonstrate the presence of an acid mucopolysaccharide surface coat in animal cells, viz. Alcian blue, colloidal iron, lanthanum, thorium, silver, SO,-coriphosphine and phosphotungstic acid. This suggests that the composition of the coat substances in the ferns and bryophytes agrees with that in animal cells. It has been found that thorium-staining in the sporocyte and the coat of the young spore of one fern, Botrychium lunaria , can be abolished with neuraminidase, indicating that sialic acid is a principal component of these coats. The spore wall in the pteridophytes (and probably also in the bryophytes) is constructed within these mucopolysaccharide surface coats, and it is suggested that differences in wall form are attributable to qualitative, quantitative and functional differences of the coats.     

附生蕨类植物的克隆性研究进展

石松类及蕨类植物在高等植物中处于比较特殊的进化与系统发育地位, 同时具有孢子植物(孢子)与种子植物(维管束)的双重特征。附生蕨类植物是蕨类植物中占据独特生境的一个大类群, 其生活史策略及进化历史与其附生生长的森林生态系统紧密相关。大部分附生蕨类植物的克隆生长习性及克隆生活史性状在其生态适应中具有重要作用, 但这方面未引起广泛关注。本文主要综述了中国山地森林中附生蕨类植物的根状茎克隆生长、克隆性与生态适应性、不同克隆生长方式与进化等方面, 并展望了蕨类植物克隆性在森林生态系统过程与功能中的作用, 以及今后如何将蕨类植物生态学研究与气候变化、植被恢复、土地利用变化等全球变化的主流方向进行结合。     

The Seed and Fern Spore Bank of a Recovering African Tropical Forest

Seed banks contribute to forest regeneration after disturbance, but less is known about fern spore banks, particularly in a paleotropical context. We sampled the buried seed and fern spore bank in Mabira Forest, a 300 km² forest in central Uganda, to explore the effect of time since disturbance. Soil cores (5 cm depth) were taken from 39 plots across three different classes of ‘recovery’: (1) not disturbed since 1950; (2) logged between 1950 and 1980; and (3) cleared for agriculture between 1970 and 1990 but reforested since. Plant emergence was monitored in a glasshouse. We predicted that the seed bank would reflect time since disturbance, with more pioneer species in recently disturbed stands, and that the fern spore bank would reflect stand age less closely due to greater dispersal capacity. We recorded a median 752 seeds per square meter, most of which were trees; the most abundant species was the invasive tree Broussonetia papyrifera. The fern spore bank was twice as dense, but 95 percent of fern spores were of one species, Christella parasitica. Tree seed density was significantly affected by time since disturbance with fewer seeds in the older stands. Herb seed density, fern spore density, and species richness for all groups were not significantly affected by time since disturbance. Neither seed bank nor fern spore bank closely resembled the aboveground vegetation. We compared our results to existing literature on seed banks in tropical forests, finding that our densities are relatively high for African forests, but low compared to the Neotropics and Australia.     

长白山哈泥泥炭地丘间生境泥炭藓孢子的寿命

长寿有性繁殖体对于植物种群的长存具有重要意义,迄今,泥炭地苔藓植物孢子长寿性研究还很少。在长白山哈泥泥炭地钻取丘间表层泥炭样品,测定泥炭腐殖化度和烧失量,逐层提取和培养泥炭藓孢子,研究埋藏时间对孢子萌发的影响。结果表明,丘间泥炭藓孢子埋藏环境中,随着埋深的增加即埋藏年限的增加,泥炭腐殖化度和烧失量总体上分别呈现增加和递减的趋势,而地层泥炭藓孢子萌发率呈现直线递减的规律,但在埋藏近150余年后孢子萌发率仍可达40%。研究进一步证明泥炭藓具有长期持久孢子库,根据推算,泥炭地丘间埋藏环境中,泥炭藓孢子最大寿命可超过400a。     

Cainozoic ferns and their distribution

The phytogeographic distribution of Cainozoic ferns is reported based upon a critical re-appraisal of the macrofossil and mesofossil record also taking account of evidence from a few highly diagnostic spores. Well-documented circum-Arctic Cainozoic floras show ferns (Woodwardia, Onoclea, Osmunda, Coniopteris and to a lesser extentAzolla) distributed around the pole to very high paleolatitudes. Some ferns are shared between the mid-paleolatitudes of North America and Europe as would be predicted from the distributions of other biota. Evidence for the composition of Cainozoic fern floras is minimal in some regions (e.g., Antarctica, Central and South America, Africa, India, South East Asia), so the absence of fern fossils from these areas has no biogeographical significance. Matoniaceae were abundant in the preceding Mesozoic. However, the absence of Cainozoic macrofossils, and the fact that no CainozoicMatonisporites spores areMatonia-like, indicates that Matoniaceae had attained their modern relict distribution by, or very early in, the Cainozoic. The important Mesozoic families Marattiaceae and Dipteridaceae are also not represented by Cainozoic macrofossils. They probably also showed Cainozoic restriction but spores are not sufficiently diagnostic to enable testing of this hypothesis. Other ferns, which were also important in the Mesozoic (e.g., Dicksoniaceae, Gleicheniaceae), have patchy, equivocal, or inadequately published Cainozoic records. The dispersed spore record may provide an opportunity to track Cainozoic Gleicheniaceae but this approach is not without problems. Most well-represented Cainozoic fern families, genera and subgenera show widespread Cainozoic ranges, typically with considerable range extensions over their living relatives, both onto other continents and north and south to higher paleolatitudes. These include Schizaeaceae (Lygodium, Anemia, and the extinctRuffordia), Osmundaceae (Osmunda), Pteridaceae (Acrostichum), Thelypteridaceae (Cyclosorus), Lophosoriaceae (Lophosoria), Cyatheaceae (theCnemidaria/Cyathea decurrens clade) and the heterosporous water fernAzolla (Azollaceae). A few well-represented ferns show Cainozoic distributions similar to those of the present day (e.g.,Salvinia [Salviniaceae] andCeratopteris [Pteridaceae] (the latter by the Neogene and based only on spores]) but even these had slightly broader ranges in the Cainozoic. Some Cainozoic ferns have apparently local distributions, e.g.,Blechnum dentatum (Blechnaceae) in Europe; and others are so far represented at only one or few sites, e.g.,Dennstaedtiopsis (Dennstaedtiaceae),Botrychium (Ophioglossales),Grammitis (Grammitidaceae), andMakotopteris andRumohra (Dryopteridaceae). Cainozoic fossils assigned toDryopteris (and some other dryopteroids) require revision along with those of Thelypteridaceae, the latter having high potential to provide useful paleobiogeographic evidence, at least of theCyclosorus group. Cainozoic records of Hymenophyllaceae and Polypodiaceae are here considered unconfirmed.     

STUDIES OF PALEOZOIC MARATTIALEANS: NEW SPECIES OF SCOLECOPTERIS (MARATTIALES) FROM THE PENNSYLVANIAN OF NORTH AMERICA

Two new species of the late Paleozoic fern Scolecopteris (Marattiales) are described and their relationships within the genus are discussed. Scolecopteris charma sp.n., from Steubenville, Ohio (Duquesne Coal, Upper Pennsylvanian), is similar to species in the Oliveri group, while S. gnoma sp.n. from Providence, Kentucky (Baker Coal, Middle Pennsylvanian), compares favorably with the Latifolia species group. Scolecopteris gnoma is most similar to S. fragilis but differs in its smaller synangia and spore type. S. charma appears generally similar to S. iowensis because of its large pedicel and histologically undifferentiated walls, but differs in a number of characters such as vasculature and spore type. Despite its occurrence late in the Pennsylvanian, S. charma is thought to possess a number of primitive character states (large trilete spores, vascularized pedicels, flat pinnules with downturned margins). Using the same criteria for the much older S. gnoma, we note a number of relatively apomorphic character states (small monolete spores, unvascularized pedicels, extended pinnule margins). An outgroup analysis of species-level characters of Scolecopteris gives a better concept of primitive versus derived traits in marattialean and other ferns. Genera in the Paleozoic fern orders Filicales (Ankyropteris) and Zygopteridales (Corynepteris, Musatea) were chosen as outgroups, and the comparisons support suggestions for the polarity of several important characters. Some of these agree with previously proposed evolutionary polarities based on the geological occurrence of marattialean ferns.